Method for removal of viruses from blood by lectin affinity hemodialysis

ABSTRACT

The present invention relates to a method for using lectins that bind to pathogens having high mannose surface glycoproteins or fragments thereof which contain high mannose glycoproteins, to remove them from infected blood or plasma in an extracorporeal setting. Accordingly, the present invention provides a method for reducing viral load in an individual comprising the steps of obtaining blood or plasma from the individual, passing the blood or plasma through a porous hollow fiber membrane wherein lectin molecules are immobilized within the porous exterior portion of the membrane, collecting pass-through blood or plasma and reinfusing the pass-through blood or plasma into the individual.

This application is a continuation of U.S. patent application Ser. No.10/760,810, filed Jan. 20, 2004, which claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 60/440,771, filed Jan. 17,2003, the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic methodologiesfor treating viral infections.

BACKGROUND OF THE INVENTION

A large number of viruses have been described which are pathogenic forhumans. Among these viruses are many for which neither drugs norvaccines are available. In cases where drug treatments are available,the occurrence of resistant mutations and drug side effects often limitthe effectiveness of therapy. Examples of such viruses include HepatitisC and human immunodeficiency virus (HIV).

HIV is the etiological agent of acquired immunodeficiency syndrome(AIDS). It infects selected cells of the immune system therebycompromising the infected individual's immune response. It is estimatedthat there are over 1 million HIV infected individuals in the UnitedStates and over 13 million worldwide. The clinical course of HIVinfection typically consists of a prolonged asymptomatic state, followedby a depletion of T4 lymphocytes making the individual susceptible toopportunistic infections and neoplasms.

HIV-1 replication occurs predominantly in CD4+ lymphocytes, the majorityof which are located in lymphoid organs, such as peripheral lymph nodesand spleen. HIV-1 can also be found in macrophages and macrophage-likecells, such as microglia in the central nervous system (Cohen et al.Immunol Rev 159: 31-48, 1997).

Plasma HIV-1 levels and presence of HIV-1 infected lymphocytes inperipheral blood strongly correlate with the clinical status of HIV-1infected patients (Ferre et al. J Acquir Immune Defic Syndr HumRetrovirol 10(Suppl 2): S51-56, 1995; O'Brien et al. N Engl J Med334(7): 426-431, 1996). The half-life of circulating virions is 6 hours,while the half-life of HIV-1 infected cells in peripheral blood is 1.6days. Greater than 10¹⁰ virions may be released into the circulationeach day (Ho et al. J Biol Regul Homeost Agents 9(3): 76-77, 1995; Ho etal. Nature 373(6510): 123-126, 1995; Wei et al. Nature 373(6510):117-122, 1995), The ability of the host immune system to keep HIVinfection in check, and limit clinical symptoms, is directlyproportional to the viral burden. Anti-retroviral therapies, nucleosideanalogues, non-nucleoside reverse transcriptase inhibitors, and proteaseinhibitors, aim to reduce the viral burden so that the immune system cancontrol or clear residual infection (Fauci, Harrisons Principles ofInternal Medicine: 1791-1856, 1998).

HIV infection is mediated by gp120, which binds to CD4 as well as to asurface chemokine receptor. Inside the cell the virion is uncoated andthe viral RNA is reverse transcribed into double-stranded DNA. ProviralDNA enters the cell nucleus, integrates into the host genome and istranscribed into viral RNAs, which are translated into viral proteins.Mature virions are assembled and released from the cell by budding.(Fauci et al. Ann Intern Med 124(7): 654-663, 1996). A dying cell mayalso release all its contents including intact virions, and fragmentsthereof into the blood. Thus, circulating blood of HIV-infectedindividuals contains intact virions, and viral proteins, in particulartoxic viral surface proteins.

The hallmark of AIDS is the gradual loss of CD4+ T cells, whichultimately leaves the immune system unable to defend againstopportunistic infections. While the mechanism through which HIV causesAIDS is imperfectly understood, the clinical data suggest that inaddition to the loss of infected T-cells, a large number of uninfectedT-cells are dying and that HIV derived envelope proteins appear to beintimately involved.

The major HIV envelope glycoprotein gp120 has been shown to haveprofound biological effects in vitro. Gp120 causes CD4+ T cells toundergo apoptosis and binding of gp120 to CD4+ cells in the presence ofanti-envelope antibodies and complement opsoninizes the cells, targetingthem for clearance. The combined effect is the destruction of uninfectedimmune cells. In addition, HIV envelope proteins have been implicated inHIV related hyper-gammaglobulinemia. In AIDS patients, gp120 levels havebeen measured at an average of 29 ng/ml which is orders of magnitudehigher than the concentration of the virus.

Currently there is no cure for HIV infection. Reverse transcriptase andprotease inhibitors have been approved for the treatment of HIV. Typicaltreatment regimes use combinations of approved drugs and are termedHAART (highly active antiretroviral therapy). While more than.16 drugsand drug combinations have been approved by the FDA for treating HIVinfection, the emergence of drug resistant mutants and the presence ofthe untreatable virus reservoirs (e.g. in memory T cells) has limitedtheir usefulness. Unfortunately, no effective HIV vaccine has beenforthcoming due, in part, to the rapid mutation of the HIV genome andthe inaccessibility of immunogenic epitopes of viral proteins. Thusthere is an urgent need for new treatments.

Extracorporeal treatments provide a therapeutic modality which may beused to treat systemic disease. Extracorporeal perfusion of plasma overprotein A, plasmapheresis and lymphapheresis have all been used asimmunomodulatory treatments for HIV infection, and the thrombocytopeniaresulting from it (Kiprov et al. Curr Stud Hematol Blood Transfus 57:184-197, 1990; Mittelman et al. Semin Hematol 26(2 Suppl 1): 15-18,1989; Snyder et al. Semin Hematol 26(2 Suppl 1): 31-41, 1989; Snyder etal. Aids 5(10): 1257-1260, 1991). These therapies are all proposed towork by removing immune complexes and other humoral mediators, which aregenerated during HIV infection. They do not directly remove HIV virus.Extracorporeal photopheresis has been tested in preliminary trials as amechanism to limit viral replication (Bisaccia et al. J Acquir ImmuneDefic Syndr 6(4): 386-392, 1993; Bisaccia et al. Ann Intern Med 113(4):270-275, 1990). However, none of these treatments effectively removeboth virus and viral proteins.

Chromatographic techniques for the removal of HIV from blood productshave been proposed. In 1997, Motomura et al., proposed salts of asulfonated porous ion exchanger for removing HIV and related substancesfrom body fluids (U.S. Pat. No. 5,667,684). Takashima and coworkers(U.S. Pat. No. 5,041,079) provide ion exchange agents comprising a solidsubstance with a weakly acidic or weakly alkaline surface forextracorporeal removal of HIV from the body fluids of a patient. Bothare similar to the work of Porath and Janson (U.S. Pat. No. 3,925,152)who described a method of separating a mixture of charged colloidalparticles, e.g. virus variants by passing the mixture over an adsorbentconstituted of an insoluble, organic polymer containing amphotericsubstituents composed of both basic nitrogen-containing groups andacidic carboxylate or sulphonate groups (U.S. Pat. No. 3,925,152).However, none of these chromatographic materials are selective forviruses and will clearly remove many other essential substances. Thusthey are not useful for in vivo blood purification.

Immunosorptive techniques have also been proposed for the treatment ofviral infections. In 1980, Terman et al. described a plasmapheresisapparatus for the extracorporeal treatment of disease including a devicehaving an immunoadsorbent fixed on a large surface area spiral membraneto remove disease agents (U.S. Pat. No. 4,215,688). The deviceenvisioned no method for directly treating blood and required thepresence of an immunologically reactive toxic agent. In 1987 and 1988,Ambrus and Horvath described a blood purification system based onantibody or antigen capture matrices incorporated onto the outsidesurface of an asymmetric, toxin permeable membrane (U.S. Pat. Nos.4,714,556; 4,787,974), however, no examples of pathogen removal weregiven therein. In 1991, Lopukhin et al. reported that rabbit antiseraraised against HIV proteins, when coupled to Sepharose 4B or silica,could be used for extracorporeal removal of HIV proteins from the bloodof rabbits which had been injected with recombinant HIV proteins(Lopukhin et al. Vestn Akad Med Nauk SSSR 11: 60-63, 1991). However,this strategy was inefficient as it required extracorporeal absorptionof blood and did not provide for a mechanism to remove free HIV viralparticles from the blood (Lopukhin et al., 1991, supra). U.S. Pat. No.6,528,057 describes the removal of virus and viral nucleic acids usingantibodies and antisense DNA.

Lectins are proteins that bind selectively to polysaccharides andglycoproteins and are widely distributed in plants and animals. Althoughmany are insufficiently specific to be useful, it has recently beenfound that certain lectins are highly selective for enveloped viruses(De Clercq. et al Med Res Rev 20(5): 323-349, 2000). Among lectins whichhave this property are those derived from Galanthus nivalis in the formof Galanthus nivalis agglutinin (“GNA”), Narcissus pseudonarcissus inthe form of Narcissus pseudonarcissus agglutinin (“NPA”) and a lectinderived from blue green algae Nostoc ellipsosporum called “cyanovirin”(Boyd et al. Antimicrob Agents Chemother 41(7): 1521-1530, 1997; Hammaret al. Ann N Y Acad Sci 724: 166-169, 1994; Kaku et al. Arch BiochemBiophys 279(2): 298-304, 1990). GNA is non-toxic and sufficiently safethat it has been incorporated into genetically engineered rice andpotatoes (Bell et al. Transgenic Res 10(1): 35-42, 2001; Rao et al.Plant 15(4): 469-477, 1998). These lectins bind to glycoproteins havinga high mannose content such as found in HIV surface proteins (Chervenaket al. Biochemistry 34(16): 5685-5695, 1995). GNA has been employed inELISA to assay HIV gp120 in human plasma (Hinkula et al. J ImmunolMethods 175(1): 37-46, 1994; Mahmood et al. J Immunol Methods 151(1-2):9-13, 1992; Sibille et al. Vet Microbiol 45(2-3): 259-267, 1995) andfeline immunodeficiency virus (FIV) envelope protein in serum (Sibilleet al. Vet Microbiol 45(2-3): 259-267, 1995). While GNA binds toenvelope glycoproteins from HIV (types 1 and 2), simian immunodeficiencyvirus (SIV) (Gilljam et al. AIDS Res Hum Retroviruses 9(5): 431-438,1993) and inhibits the growth of pathogens in culture, (Amin et al.Apmis 103(10): 714-720, 1995; Hammar et al. AIDS Res Hum Retroviruses11(1): 87-95, 1995) such in vitro studies do not reflect the complex,proteinacious milieu found in HIV infected blood samples. It istherefore not known if lectins capable of binding high mannoseglycoproteins in vitro would be able to bind such molecules in HIVinfected blood samples. On the contrary, it is generally considered thatthe high concentrations of antibodies to gp120 typically present inindividuals infected with HIV would sequester the high mannoseglycoprotein sites to which lectins such as GNA bind.

Accordingly, although lectins are known to bind viral envelopeglycoproteins, no previous technologies have been developed usinglectins to directly adsorb HIV or other enveloped viruses from the bloodusing in vivo dialysis or plasmapheresis. Therefore, there is an ongoingneed for novel therapeutic approaches to the treatment of HIV and otherviral infections. In particular, there is a need for the development ofnovel approaches to reduce viral load so as to increase theeffectiveness of other treatments and/or the immune response.

SUMMARY OF THE INVENTION

The present invention relates to a method for using lectins that bind topathogens having high mannose glycoproteins or fragments thereof toremove them from infected blood or plasma in an extracorporeal setting.Accordingly, the present invention provides a method for reducing viralload in an individual comprising the steps of obtaining blood or plasmafrom the individual, passing the blood or plasma through a porous hollowfiber membrane wherein lectin molecules are immobilized within theporous exterior portion of the membrane, collecting pass-through bloodor plasma and reinfusing the pass-through blood or plasma into theindividual.

Passage of the blood through the hollow fibers having immobilized lectincauses the virions and fragments thereof which contain high mannoseglycoproteins to bind to the lectins, thereby reducing the viral load inthe effluent. In one embodiment, this invention uses lectins that bindviral envelope proteins of many subtypes of HIV types 1 and 2 and SIV.The method of the present invention reduces the number of virions in theblood and rapidly and effectively reduces the levels of viral surfaceproteins in infected blood which may be toxic. It will be apparent tothose skilled in the art that the method will assist in the clearance ofother infections, frequently occurring simultaneously with HIV-1, suchas hepatitis C virus (HCV) (Fauci et al., 1998, supra).

Thus, an object of the invention is to provide a method for reducing theviral load in the blood of an individual infected with a virus. In oneembodiment, virions or protein fragments thereof or combinations thereofare removed from the blood of an individual infected with a virus.

Another object of the present invention is to provide a method forreducing the viral load in the blood by extracorporeal circulation ofblood through hollow fibers containing immobilized lectins havingaffinity for viral high mannose glycoproteins.

Another object of the present invention is to provide an apparatuscomprising hollow fibers, wherein the exterior surface of the fibers isin close proximity with immobilized lectins having specific affinity forhigh mannose glycoproteins in the virus or other pathogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a longitudinal cross section of anaffinity cartridge.

FIG. 2 is a schematic illustration of a horizontal cross section atplane 2 in FIG. 1.

FIG. 3 is an illustration of a channel from FIG. 2. A hollow fibermembrane structure 40 is composed of a tubular section comprising arelatively tight ultrafiltration membrane 42 and relatively porousexterior portion 44 in which may be immobilized affinity molecules 46,such as lectins.

FIG. 4 is a graphical representation of the removal of gp120 from HIVloaded physiological saline. Initial gp120 was 500 ng/ml in PBS (1.6ml/run). Gp120 was recirculated over a column containing 0.2 ml GNAagarose vs Sepharose 4B control at 0.5-0.58 ml/min and room temperature.

FIG. 5 is a graphical representation of the removal of gp120 immunecomplexes from HIV infected human plasma. Initial gp120 was 500 ng/ml inhuman HIV+ plasma (1.6 ml/run). Assayed with 10 μg/well GNA/NPA plate tocapture gp120 immune complexes detected with sheep anti-human IgG.Plasma was recirculated over a Glen Research column containing 0.2 mlGNA agarose vs Sepharose 4B control at 0.5-0.58 ml/min and roomtemperature. Lines are theoretical exponential best-fit R²=0.91 forExperimental (ο) and linear for Control (□).

FIGS. 6A and 6B demonstrate the removal of native HIV on GNA AgaroseFIG. 6A is a graphical representation of a plasmapheresis exponentialcurve where R²=0.90 (excluding one point at 22 hours). FIG. 6B is agraphical representation of a log plot of initial removal rate, wherehalf time ˜0.9 hours. Conditions Masterflex pump with #14 silicon tubing(1.1 ml/min). Plasma sample 3 ml initial volume (100,000 copies per ml(CPM) BBI ER8-03030-0002 native HIV). Aliquot volume was 250 ul plasmafor RNA isolation. Realtime RTPCR with Sybr green tracking dye.Thermocycling 95, 60, 72, 83° C. (15, 30, 60 sec, read 6 sec). Ctcalculated from the primary curve at T=20.

FIG. 7 is a graphical representation of the removal of gp120 from HIV⁺blood. Initial gp120 was 100 ng/ml in human HIV⁺ plasma. Assayed with0.1 ug/well GNA-NPA plate with immune complexes disrupted withacid/detergent prior to assay. The blood was recirculated over aMicrokros column containing 0.6 ml GNA agarose vs. Sepharose 4B control.Flow rate 0.9 ml/min at 37° C. using a Masterflex pump (1 rpm) andPharmed 6485-16 tubing. Lines are theoretical exponential best-fitR²=0.91 for Experimental (□) (t_(1/2)=22 min) and linear for Control(ο).

FIG. 8 is a graphical representation of the the removal of Hepatitis Cvirus infected blood. The blood was recirculated over a Microkros columncontaining 0.6 ml GNA agarose vs. Sepharose 4B control. Flow rate 0.5ml/min at room temperature using a Masterflex pump (1 rpm) and Pharmed6485-16 tubing. The line is a theoretical exponential best-fit R²=0.85.

DETAILED DESCRIPTION OF THE INVENTION

The term “viral load” as used herein for the purpose of specificationand claims refers to the amount of viral particles or toxic fragmentsthereof in a biological fluid, such as blood or plasma. Viral load isaccordingly related to the number of virus particles in the body. Viralload can therefore be a measure of any of a variety of indicators of thepresence of a virus, such as viral copy number per unit of blood orplasma or units of viral proteins or fragments thereof per unit of bloodor plasma.

The term “high mannose glycoprotein” as used herein for the purpose ofthe specification and claims refers to glycoproteins havingmannose-mannose linkages in the form of α-1→3 or α-1→6 mannose-mannoselinkages. Some examples of such lectins include GNA, NPA, cyanovirin andConconavalin A (ConA).

The present invention relates to a method for using lectins to removepathogenic organisms and fragments thereof from infected blood or plasmain an extracorporeal setting. Accordingly, the present inventionprovides a method for reducing viral load in an individual comprisingthe steps of obtaining blood or plasma from the individual, passing theblood or plasma through a porous hollow fiber membrane wherein lectinmolecules which bind to high mannose glycoproteins are immobilizedwithin the porous exterior portion of the membrane, collectingpass-through blood or plasma, and reinfusing the pass-through blood orplasma into the individual.

In a preferred embodiment, the method of the present invention iscarried out by using an affinity cartridge using the device illustratedin FIG. 1. Devices of this general type are disclosed in U.S. Pat. Nos.4,714,556, 4,787,974 and 6,528,057, the disclosures of which areincorporated herein by reference. In this device, blood is passedthrough the lumen of a hollow fiber ultrafiltration membrane that is inintimate contact, on the non-blood wetted side of the membrane, withimmobilized lectins, which form a means to accept and immobilize virusesand toxic and/or infectious fragments thereof. Thus, the device retainsintact virions and viral glycoproteins bound by lectin while allowingother components to pass through the lumen.

HIV is the prototypic virus for which this invention is described, butthe invention can be adapted to the removal of any blood-borne viruses.The device, described in detail in FIGS. 1-3 includes multiple channelsof hollow fiber ultrafiltration membrane that forms a filtrationchamber. An inlet port and an effluent port are in communication withthe filtration chamber. The ultrafiltration membrane is preferably ananisotropic membrane with the tight or retention side facing thebloodstream. The membrane is conveniently formed of any number ofpolymers known to the art, for example, polysulfone, polyethersulfone,polyamides, polyimides, cellulose acetate, and polyacrylamide.Preferably, the membrane has pores 200-500 nm in diameter, which willallow passage of intact viruses and viral particles and fragments (e.g.,HIV virions of 110 nm diameter), but not most blood cells (red bloodcells 2,000 nm diameter, lymphocytes 7,000-12,000 nm diameter,macrophages 10,000-18,000 nm diameter). A diagram of the device is shownin FIG. 1. The device comprises a cartridge 10 comprising ablood-processing chamber 12 formed of interior glass wail 14. Aroundchamber 12 is an optional exterior chamber 16. A temperature controllingfluid can be circulated into chamber 16 through port 18 and out of port20. The device includes an inlet port 32 for the blood and an outletport 34 for the effluent. The device also provides one or more ports 48and 50, for accessing the extrachannel space in the cartridge. As shownin FIGS. 1 and 2, chamber 12 contains a plurality of ultrafiltrationmembranes 22. These membranes preferably have a 0.3 mm inside diameterand 0.5 mm outside diameter. FIG. 3 is a cross sectional representationof a channel 22 and shows the anisotropic nature of the membrane. Asshown in FIG. 3, a hollow fiber membrane structure 40 is composed of asingle polymeric material which is formed into a tubular sectioncomprising a relatively tight ultrafiltration membrane 42 and relativelyporous exterior portion 44 in which may be immobilized lectins 46.During the operation of the device, a solution containing the lectins isloaded on to the device through port 48. The lectins are allowed toimmobilize to the exterior 22 of the membrane in FIG. 2. Unbound lectinscan be collected from port 50 by washing with saline or other solutions.

For the method of the present invention, blood having viral particlesand/or fragments thereof is withdrawn from a patient and contacted withan ultrafiltration membrane. In one preferred embodiment, the blood isseparated into its plasma and cellular components. The plasma is thencontacted with the lectins to remove the viral particles or fragmentsthereof by binding between viral high mannose glycoproteins and lectins.The plasma can then be recombined with the cellular components andreturned to the patient. Alternatively, the cellular components may bereturned to the patient separately. The treatment can be repeatedperiodically until a desired response has been achieved. For example,the treatment can be carried out for 4 hours once a week.

The technology to immobilize enzymes, chelators, and antibodies indialysis-like cartridges has been developed (Ambrus et al. Science201(4358): 837-839, 1978; Ambrus et al. Ann Intern Med 106(4): 531-537,1987; Kalghatgi et al. Res Commun Chem Pathol Pharmacol 27(3): 551-561,1980) and is incorporated herein by reference. These cartridges can bedirectly perfused with blood from patients through direct venous access,and returned to the patients without further manipulations.Alternatively, blood can be separated into plasma and cellularcomponents by standard techniques. The cellular components may becombined with the plasma before reinfusing or the cellular componentscan be reinfused separately. Viral load can be assessed in the effluentfrom the cartridge by standard techniques such as ELISA and nucleic acidamplification and detection techniques. Prototypic cartridges have beenused to metabolize excess phenylalanine (Kalghatgi et al., 1980, supra;Ambrus, 1978, supra) or to remove excess aluminum from patients' blood(Anthone at al. J Amer Sac Nephrol 6: 1271-1277, 1995). An illustrationof preparing proteins for immobilization to the hollow fibers for themethod of the present invention is presented in U.S. Pat. Nos. 4,714,556and 4,787,974, 5,528,057.

For binding of lectins to the ultrafiltration membrane, the polymers ofthe ultrafiltration membrane are first activated, i.e., made susceptiblefor combining chemically with proteins, by using processes known in theart. Any number of different polymers can be used. To obtain a reactivepolyacrylic acid polymer, for example, carbodiimides can be used (Valuevet al., 1998, Biomaterials, 19:41-3). Once the polymer has beenactivated, the lectins can be attached directly or via a linker to formin either case an affinity matrix. Suitable linkers include, but are notlimited to, avidin, strepavidin, biotin, protein A, and protein G. Thelectins may also be directly bound to the polymer of the ultrafiltrationmembrane using coupling agents such as bifunctional reagents, or may beindirectly bound. In a preferred embodiment, GNA covalently coupled toagarose can be used to form an affinity matrix.

The following examples are presented to illustrate this invention andare not intended to be restrictive.

EXAMPLE 1

This Example demonstrates the preparation of an affinity matrix usingGNA covalently coupled to Agarose using Cyanogen Bromide. Cyanogenbromide (CNBr) activated agarose was used for direct couplingessentially according to Cuatrecasas, et al (Cuatracasas et al. ProcNatl Acad Sci USA 61(2): 636-643, 1968). In brief, 1 ml of GNA at aconcentration of 10 mg/ml in 0.1M NaHCO₃ pH 9.5 is added to 1 ml CNBractivated agarose (Sigma, St. Louis, Mo.) and allowed to react overnightin the cold. When the reaction is complete, unreacted materials areaspirated and the lectin coupled agarose washed extensively with sterilecold PBS. The lectin agarose affinity matrix is then stored cold untilready for use. Alternatively, GNA agarose is available commercially fromVector Labs (Burlingame, Calif.)

EXAMPLE 2

This Example demonstrates preparation of the lectin affinity matrixusing GNA covalently coupled to glass beads via Schiff's base andreduction with cyanoborohydride. The silica lectin affinity matrix wasprepared by a modification of the method of Hermanson (Hermanson,Bioconjugate Techniques: 785, 1996). GNA lectin was dissolved to a finalprotein concentration of 10 mg/ml in 0.1M sodium borate pH 9.5 and addedto aldehyde derivatized silica glass beads (BioConnexant, Austin Tex.).The reaction is most efficient at alkaline pH but will go at pH 7-9 andis normally done at a 2-4 fold excess of GNA over coupling sites. Tothis mixture was added 10 μl 5M NaCNBH₃ in 1N NaOH (Aldrich, St Louis,Mo.) per ml of coupling reaction and the mixture allowed to react for 2hours at room temperature. At the end of the reaction, remainingunreacted aldehyde on the glass surfaces are capped with 20 μl 3Methanolamine pH 9.5 per ml of reaction. After 15 minutes at roomtemperature, the reaction solution was decanted and the unbound proteinsand reagents removed by washing extensively in PBS. The matrix was thestored in the refrigerator until ready for use.

EXAMPLE 3

This Example demonstrates preparation of GNA covalently coupled toaminocelite using glutaraldehyde. Aminocelite was prepared by reactionof celite (silicate containing diatomaceous earth) by overnight reactionin a 5% aqueous solution of aminopropyl triethoxysilane. The aminatedcelite was washed free of excess reagent with water and ethanol anddried overnight to yield an off white powder. One gram of the powder wasthen suspended in 5 ml 5% glutaraldehyde (Sigma) for 30 minutes. Excessglutaraldehyde was then removed by filtration and washing with wateruntil no detectable aldehyde remained in the wash using Schiff'sreagent. The filter cake was then resuspended in 5 ml of Sigmaborohydride coupling buffer containing 2-3 mg/ml GNA and the reactionallowed to proceed overnight at room temperature. At the end of thereaction, unreacted GNA was washed off and the unreacted aldehydeaminated with ethanolamine as described. After final washing in sterilePBS, the material was stored cold until ready for use.

EXAMPLE 4

This Example demonstrates the preparation of an exemplary lectinplasmapheresis device. Small volume filter cartridges (Glen Research,Silverton, Va.) were prepared containing 0.2 ml lectin resin, sealed andequilibrated with 5-10 column volumes sterile PBS. The cartridges wereused immediately.

EXAMPLE 5

This Example demonstrates preparation of a GNA lectin affinityhemodialysis device. The viral Hemopurifier was made by pumping a slurryof particulate immobilized GNA on agarose beads or celite in sterile PBSbuffer into the outside compartment of a hollow-fiber dialysis columnusing a syringe. For blood samples up to 15 mls, Microkrospolyethersulfone hollow-fiber dialysis cartridge equipped with Luerfittings (200μ ID×240μ OD, pore diameter 200-500 nm, ˜0.5 ml internalvolume) obtained from Spectrum Labs (Rancho Dominguez, Calif.) wereused. Cartridges containing the affinity resin were equilibrated with5-10 column volumes sterile PBS.

EXAMPLE 6

This Example demonstrates removal of HIV gp120 from physiological salineusing an affinity plasmapheresis device. The plasmapheresis devicedescribed in Example 4 was equilibrated with 5-10 column volumes sterilePBS. A sample ˜1.5 ml containing gp120 (typically 500 ng/ml) wascirculated over the column at a flow rate of 0.5-0.6 ml/min at roomtemperature. The circulating solution was tested at various timeintervals for the presence of gp120 and gp120 immune complexes whereappropriate.

Quantitative ELISA assays for HIV-1 gp120 were performed using amodification of the method of Weiler (Weiler et al. J Viral Methods32(2-3): 287-301, 1991). GNA/NPA plates were prepared on Greiner Cbottom plates by adding 100 ul protein (1-100 ug/ml each of GNA and NPAin PBS) to each well and incubating 2 hours at 37° C. The plates werethen washed in PBST (PBS containing 0.01% Tween 20) and blocked inCasein blocking buffer for 1 hour at 37° C. Plates not used immediatelywere stored for up to 2 weeks at 4° C.

For detection of free gp120, 100 μl samples of test solutions wereincubated for 1-2 hours at 37° C. After capture, plates were washed inPBS and 100 μl of the appropriate horse radish peroxidase (HRP) labeledanti-gp120 antibody (1:2500 in blocking buffer) was added. Afterincubation for 1 hour at 37° C. the antiserum was aspirated and theplates washed 4×300 μl PBSTA and the bound HRP detected with stabilizedtetramethylbenzidine (TMB) substrate (BioFx). For the determination ofimmune complex and immune complex formation, after capture, plates werewashed in PBS and 100 μl of affinity purified HRP labeled sheepanti-human IgG antibody (1:2500 in blocking buffer) was added. Afterincubation for 1 hour at 37° C. the antiserum was aspirated and theplates washed 4×300 μl PBSTA. Bound HRP was detected withtetramethylbenzidine (TMB) (BioFx).

FIG. 4 shows that GNA agarose removed gp120 from buffer solution with99% efficiency in <15 minutes. Because gp120 is a heavily glycosylatedprotein which can bind non-specifically to a variety of surfaces, it isnot surprising that the control column also bound 85% of the inputgp120.

EXAMPLE 7

This Example demonstrates the removal of HIV gp120 from infected plasmausing a lectin affinity plasmapheresis device. The plasmapheresis devicedescribed in Example 4 was equilibrated with 5-10 column volumes sterilePBS. A plasma sample of about 1.5 ml containing gp120 (typically 500ng/ml) was circulated over the column at a flow rate of 0.5-0.6 ml/minat room temperature. The circulating solution was tested at various timeintervals for the presence of gp120 and gp120 immune complexes whereappropriate as in Example 6.

Since anti-gp120 antibodies are typically abundant in HIV+ plasma,removal of gp120 from infected plasma might be expected to be moredifficult than removal from simple buffer solutions. In part due tothese antibodies, gp120 detection in HIV+ plasma and blood typicallyshows at best low amounts of gp120. In order to measure removal it wastherefore necessary to add gp120 to infected patient plasma to provide asample for measurement. ELISA measurement of the sample confirmed thatall of the added gp120 in this sample was complexed with anti-gp120antibodies (data not shown).

FIG. 5 shows that the GNA agarose affinity resin effectively removedgp120 in immune complexes from HIV infected plasma samples. Removal wasrapid with an apparent half reaction time of 20 minutes. A portion ofthe gp120 signal was not removed (˜10% of the initial gp120 immunecomplex) even after 7 hours and appeared to represent background bindingof IgG in the assay.

EXAMPLE 8

This Example demonstrates removal of HIV virions from infected plasmausing GNA plasmapheresis. An HIV infected plasma sample (ER8-03030-0002native HIV, Boston Biomedica, Boston Mass.) containing 100,000 copiesper ml (cpm) of the virus was circulated over a 0.2 ml GNA agarosecolumn described in Example 4. At intervals, 250 μl aliquots of theplasma were taken and the viral RNA extracted using TRI-LS reagentaccording to, the manufacturers instructions (MRC Corporation). HIVviral RNA was then quantitated using real time RT PCR and an Access 1step reagent set from Promega (Madison, Wis.) in 25 μl reaction volumescontaining 400 nM SK432 and SK461 gag gene primers, Sybr green(1:10,000), 1× SCA blocking buffer, 3 mM MgCl₂, 400 uM dNTPs and 10 ulof unknown RNA or HIV-1 RNA from armored RNA standards (Ambion AustinTex.). Amplification and reaction times were: RT (45 minutes at 48° C.)and PCR 40 cycles (94° C./15 sec; 62° C./30 sec; 72° C./60 sec; 83°C./read) in a SmartCycler real time thermocycler (Cepheid, Sunnyvale,Calif.) essentially according to the manufacturers instructions. Whennecessary for confirmation of amplification, 10 μl aliquots of theamplification mix were subjected to agarose gel electrophoresis 2%(w/v)(Sigma, molecular biology grade) in 0.5× TBE buffer pH 8.3 containing0.25 ug/ml ethidium bromide for 45 minutes at 120 VDC at roomtemperature. Gels were photographed on a UV transilluminator with theimages subsequently digitized and analyzed using ImageJ.

FIGS. 6A and 6B show that GNA agarose effectively removes HIV virionsfrom infected plasma. FIG. 6A is a linear plot of the data curve fit toa exponential decay (R²=0.9). The curve predicts essentiallyquantitative removal of HIV in about 10 hours. FIG. 6B is a log plot ofthe HIV removal rate which gives an estimate of 0.9 hours as the halftime of HIV removal. Virus removal appears first order as expected forGNA in excess over virus. CPM indicates HIV copies/ml.

EXAMPLE 9

This Example demonstrates removal of gp120 from HIV infected blood usinga GNA lectin affinity hemodialysis device. Since most HIV+ plasmasamples have low or undetectable amounts of gp120, simulated HIVinfected blood samples were prepared by mixing 5 ml type O+ fresh packedred cells with 5 ml HIV infected plasma (typically 10⁵ cpm) to which wasadded sufficient gp120 IIIB to make the sample 100 ng/ml

The affinity hemodialysis devices described in Example 5 wereequilibrated with 5-10 column volumes sterile PBS. A control columncontaining only Sepharose 4B was prepared as a control. The infectedblood sample ˜10 ml containing gp120 was recirculated over the column ata flow rate of 0.9 ml/min at 37° C. using a Masterflex roller pump (1rpm) and Pharmed 6485-16 silicon tubing. The circulating solution wastested at various time intervals for the presence of free gp120 afteracid denaturation and neutralization to disrupt immune complexes.

FIG. 7 shows that as the blood samples were recirculated over thecartridge, the initial gp120 of 100 ng/ml was reduced to backgroundlevels in 4 to 6 hours (apparent t_(1/2)=22 min). The control cartridgeremoved gp120 very slowly.

EXAMPLE 10

This example demonstrate removal of HCV from infected blood using GNAlectin affinity hemodialysis. In order to show the broad specificity ofGNA lectin removal of viruses, we performed lectin affinity hemodialysison HCV infected blood. The lectin affinity hemodialysis devicesdescribed in Example 4 were equilibrated with 5-10 column volumessterile. PBS. HCV infected blood samples were prepared by mixing 1 mltype O+ fresh packed red cells with 1 ml HIV infected plasma (typically10⁵ cpm). The infected blood sample was recirculated over the column ata flow rate of 0.5 ml/min at room temperature using a Masterflex rollerpump (1 rpm) and Pharmed 6485-16 tubing. The circulating solution wastested at various time intervals for the presence HCV viral RNA.

Viral RNA was isolated using TRI-LS (MRC Corporation) from 100 μl ofplasma according to the manufacturers instructions. HCV viral RNA wasthen measured by quantitative RT PCR performed using an ImpromII reagentset from Promega (Madison, Wis.) in 25 ul reaction volumes containing400 nM EY80 and EY78 HCV specific primers, Sybr green (1:10,000), 1× SCAblocking buffer, 3 mM MgCl₂, 400 uM dNTPs, 0.2 units/ul each of Tflpolymerase and AMV reverse transcriptase. Typically 50 ul of the mix wasused to dissolve RNA isolated from 100 μl plasma and the mix split intotwo identical duplicate samples. Amplification and reaction times were:RT (45 minutes at 48° C.) and PCR 40 cycles (94° C./15 sec; 62° C./30sec; 72° C./60 sec; 87° C. readout) in a SmartCycler real timethermocycler (Cepheid, CA) essentially according to the manufacturersinstructions. The amount of viral RNA was estimated by comparison to thesignal strength of the viral RNA standards in the initial phase of theamplification reaction (C_(t)=20).

FIG. 8 shows that as the blood was recirculated over the cartridge, theinitial HCV was reduced about 50% in 3 hours (apparent t_(1/2)=3 hours).The curve fit reasonably well to an exponential decay.

From the foregoing, it will be obvious to those skilled in the art thevarious modifications in the above-described methods, and compositionscan be made without departing from the spirit and scope of theinvention. Accordingly, the invention may be embodied in other specificforms without departing from the spirit or essential characteristicsthereof. Present examples and embodiments, therefore, are to beconsidered in all respects as illustrative and not restrictive, and allchanges which come within the meaning and range of equivalency of theclaims are therefore intended to be embraced therein.

1-68. (canceled)
 69. A method of depleting high mannose glycoproteinsfrom a sample, comprising: a) obtaining a sample comprising high mannoseglycoproteins; b) passing the sample through a porous hollow fibermembrane, wherein a lectin is immobilized within a porous exteriorportion of the porous hollow fiber membrane, and wherein the lectinbinds to high mannose glycoproteins; and c) collecting the sample after(b).
 70. The method of claim 69, wherein the sample is blood or plasma.71. The method of claim 70, wherein the blood or plasma is obtained froman individual, and further comprising reinfusing the pass-through bloodor plasma into the individual.
 72. The method of claim 69, wherein thelectin is selected from the group consisting of Galanthus nivalisagglutinin (GNA), Narcissus pseudonarcissus agglutinin (NPA),cyanovirin, and Conconavalin A and mixtures thereof.
 73. The method ofclaim 72, wherein the lectin is GNA.
 74. A method of isolating highmannose glycoproteins from a sample, comprising: passing a samplecomprising high mannose glycoproteins through a porous hollow fibermembrane, which comprises a lectin immobilized within a porous exteriorportion of the porous hollow fiber membrane, wherein the lectin binds tothe high mannose glycoproteins thereby isolating high mannoseglycoproteins within the porous hollow fiber membrane.
 75. The method ofclaim 74, wherein the sample is blood or plasma.
 76. The method of claim74, wherein the lectin is selected from the group consisting ofGalanthus nivalis agglutinin (GNA), Narcissus pseudonarcissus agglutinin(NPA), cyanovirin, and Conconavalin A and mixtures thereof.
 77. Themethod of claim 76, wherein the lectin is GNA.
 78. A method ofquantifying an amount of high mannose glycoproteins in a sample,comprising: a) passing a sample, which comprises an amount of highmannose glycoproteins, through a porous hollow fiber membrane, whichcomprises a lectin immobilized within a porous exterior portion of theporous hollow fiber membrane, wherein the lectin binds to the highmannose glycoproteins in the sample; and b) quantifying an amount ofhigh mannose glycoproteins in the sample after (a).
 79. The method ofclaim 78, wherein the sample is blood or plasma.
 80. The method of claim78, wherein the lectin is selected from the group consisting ofGalanthus nivalis agglutinin (GNA), Narcissus pseudonarcissus agglutinin(NPA), cyanovirin, and Conconavalin A and mixtures thereof.
 81. Themethod of claim 80, wherein the lectin is GNA.